1) Molecular machinery regulating protein secretion in the acinar cells of salivary glands (33% effort) [unreadable] The major secretory units in the salivary glands are the acini that are formed by pyramidal polarized cells, which form a small lumen where salivary proteins and water are secreted. Proteins destined to secretion are packed in secretory granules in the trans-Golgi network (TGN) from where they are transported through cytoplasm to the cell periphery. Here, upon stimulation of either muscarinic or adrenergic receptors, the granules fuse with the apical domain of the plasma membrane (PM) releasing their content into the apical lumen. [unreadable] Our aim is to study the molecular machinery regulating the formation of the granules at the TGN and their fusion with the PM. Due the lack of a reliable cell culture model these machinery in the salivary glands are still largely unknown, while in other exocrine glands, like the pancreas, substantial progress has been made. [unreadable] We plan to study these mechanisms by using three different and complementary model systems and namely, live animals, three-dimensional (3D) cell culture systems and organ explants.[unreadable] Establishing an animal model to study regulated protein secretion in mice[unreadable] To study protein secretion in live animals by intra-vital multi-photon, we are planning to engineer mice to express fluorescently labeled proteins that localize to the secretory granules. Our aim is to follow in real time the agonist-stimulated release of the granules in control conditions or when specific genes of interest have been ablated via viral mediated expression of short hairpin RNA (shRNA). To this aim we have selected candidate proteins and their genes were fused with various fluorescent tags either at the C- or the N- termini. The addition of the tag did not affect their localization and did not induce any cellular toxicity as established by expressing the constructs both transiently and stably in either 2D or 3D cultured cells. We are now in the process of transducing these genes in the submandibular glands of live mice and rats in order to determine whether these molecules are properly targeted to the secretory granules and whether they affect regulated protein secretion in vivo.[unreadable] Establishing a three-dimensional cell culture system to study the mechanisms of protein secretion[unreadable] Cells derived from the intercalated ducts of human sub-mandibular glands (HSG) are believed to differentiate into acini, ducts, or myoepithelial cells depending on the growth conditions. While HSG cells grown on plastic or glass do not express any of the secretory markers characteristic of the differentiated salivary glands cells, and do not show any polarization, remarkably, when grown on basement membranes they assume an acinar phenotype and express secretory proteins. However, it was not established whether these cells are competent to secrete cargo proteins and whether they have established polarized domains. We have shown that these 3D-grown HSG cells are not fully differentiated since they express both myoepithelial and acinar markers and furthermore, they are only partially polarized since only some molecules are properly targeted to their correct domain of the plasma membrane. We have also observed that growing the cells on a substrate mimicking the basal membrane promotes the activation of the sorting machinery at the TGN but that the cells are not still competent to secrete. These finding suggest that key component other than the basal membranes are necessary for the complete differentiation of the HSG cells into fully functional secretory units and we are currently investigating for these factors. [unreadable] 2) Molecular machinery regulating the sorting of proteins from the trans-Golgi network to the apical or the basolateral domain of the plasma membrane (33% effort)[unreadable] Proteins are sorted from the TGN to the apical or the basolateral domain of the PM. Whether these proteins follow a direct route to the PM or they are first diverted towards other intracellular compartments is still a matter of debate. Furthermore, the molecular machineries regulating the sorting events are not still understood. We are focusing our attention on two proteins belonging to a family of water channels expressed in acinar cells, aquaporin 3 (AQP3) and aquaporin 5 (AQP5), which are localized to the basolateral and the apical domain respectively. Our strategy is to express in live animals fluorescently tagged AQP3 and AQP5 by using viral-based expression vectors and to assess by time lapse imaging the route followed by these proteins once they are released from the TGN. We have already cloned both proteins from human, rats and mice, tagged them with various fluorescent molecules such as YFP, m-cherry and PA-GFP, and introduced various mutations in their cytoplasmic tails. The analysis of their distribution and trafficking in the live animal is in progress.[unreadable] 3) Molecular machinery regulating endocytosis in salivary glands of live animals (33% effort) [unreadable] The role of endocytosis in the physiology of the salivary glands has never been elucidated. Uptake of proteins from both the apical and the basolateral domains of the ductal system have been described but never thoroughly investigated. The presence under physiological conditions of salivary proteins in the bloodstream and of serum proteins in the saliva, argues strongly in favor of a constant and bi-directional transcytotic movement of proteins across the salivary gland epithelium. The salivary glands offer a unique opportunity to study endocytosis in polarized epithelial cells in a physiological context since both the basolateral and the apical domains are accessible from the bloodstream and from the major excretory duct, respectively. [unreadable] Our aim is to define the endocytic pathways in the salivary glands, to investigate the molecular machinery regulating the internalization of various cargo with a particular emphasis on the role of the cytoskeleton, and finally to understand what is the contribution of the endocytic events in the physiology of the glands and especially during secretion. [unreadable] Characterization of the endocytic pathways in rat submandiular glands of live rodents[unreadable] We set up an experimental system to study endocytosis and transcytosis in live rodents by intra-vital TPM. First, we showed that taking advantage of the unique properties of the TPM, the architecture of the salivary glands can be visualized in details analyzing at the same time the parenchyma of the tissue, the extracellular matrix, and a broad spectrum of fluorescent probes that are administered exogenously. Next, we showed that systemically injected probes such as dextrans, bovine serum albumin (BSA), and transferrin can be internalized primarily by the fibroblasts present in the stroma of the glands. We showed that the internalization of these probes occurs within the first few minutes and that their trafficking from the early to late endosomal/lysosomal compartments can be followed dynamically in the live animal. Notably, we were able for the first time to image fusion events at a resolution comparable to that achieved in cell culture by confocal microscopy. This enabled us to unravel that later stage of delivering molecules to the lysosomes occurs through a mixed process of maturation and transient interconnections. We also showed that fluorescent probes can be administered though the Whartons duct, filling the entire ductal systems and opening new avenues to study endocytosis from the apical domains of the salivary glands. Notably, the same route can be utilized to specifically deliver pharmacological agents avoiding thus systemic injections. As proof of principle we showed that drugs such as cytochalasin D and latrunculin A, which disrupt the actin cytoskeleton, induce a reduction in the internalization of the injected probes.